A number of circulating micro-ribonucleic acids (miRNAs) have been introduced as convincing predictive determinants in a variety of cardiovascular diseases. This study aimed to evaluate some miRNAs’ diagnostic and prognostic value in patients with acute heart failure (AHF).Forty-four AHF patients were randomly selected from a tertiary heart center, and 44 healthy participants were included in the control group. Plasma levels of assessed miRNAs, including miR -1, -21, -23, and -423-5-p were measured in both groups. The patients were followed for one year, and several clinical outcomes, including in-hospital mortality, one-year mortality, and the number of readmissions, were recorded.An overall 88 plasma samples were evaluated. There was no significant difference in terms of demographic characteristics between the AHF and healthy groups. Our findings revealed that mean levels of miR-1, -21, -23, and -423-5-p in AHF patients were significantly higher than in the control group. Although all assessed miRNAs demonstrated high diagnostic potential, the highest sensitivity (77.2%) and specificity (97.7%) is related to miR-1 for the values above 1.22 . Besides, the levels of miR-21 and -23 were significantly lower in patients with ischemia-induced HF. However, the follow-up data demonstrated no significant association between miRNAs and prognostic outcomes including in-hospital mortality, one-year mortality, and the number of readmissions.The result of our study demonstrated that miR-1, -21, -23, and -423-5-p can be taken into account as diagnostic aids for AHF. Nevertheless, there was no evidence supporting the efficacy of these miRNAs as prognostic factors in our study. Acute heart failure (AHF) is one of the most life-threatening and burdensome cardiovascular diseases (CVD) in both developed and developing countries , 2. The Despite well-established biomarkers, including brain (B-type) natriuretic peptide (BNP) and N-terminal pro-BNP (NT-proBNP) for assigning the diagnosis of AHF, there are some limitations in this era to apply as reliable indicators. For instance, a series of patient characteristics and conditions such as older age, obesity, renal dysfunction, atrial fibrillation, thromboembolic events, etc., can affect serum levels of BNP/ NT-proBNP and may lead to false results , 9. MoreMicro-RNAs (miRNAs) are small non-coding RNAs with 21-25-nucleotide to regulate various genes . Since tMiR-1 is one of the most abundant miRNAs in cardiac tissue and is linked to cardiac remodeling being inversely correlated with hypertrophy. Experimental animal studies showed that adenoviral delivery of miR-1 was able to reverse hypertrophy , 20. It MiR-21 plays a role in inflammation and smooth muscle proliferation and fibrosis. It has been found to be increased in mice hearts subjected to ischemia-reperfusion and more specifically in fibroblasts within the infarct zone , 21. It MiR-23 is upregulated in cardiac hypertrophy and promotes hypertrophy, while antagomiR-mediated silencing of miR-23a reverses it , 21.Finally, miR-423-5-p has been also found to be increased in HF. In a small study, Tijsen et al. showed that miR-423-5-p strongly discriminated HF-associated dyspnea from healthy controls (C-statistic 0.91) and non-HF dyspnea (C-statistic 0.83) and was correlated with NT-proBNP and LVEF . In anotOn the role of miRNAs in HF prognosis, a very recent systematic review and meta-analysis showed that HF patients with low expression of miR-1, miR-21, miR-23, and miR-423-5-p have significantly worse overall survival. However, among them, miR-423-5-p is the stronger biomarker for HF prognosis .Many experimental animal, preclinical, and clinical human studies and reviews have tried to explore the role of miR in HF diagnosis with much attention focused on their relative superiority or non-inferiority compared to established biomarkers such as BNP or NT-proBNP , 21. MicS1 Fig).This study consisted of two phases. In the first phase, the plasma samples of the AHF patients and the healthy control group were evaluated to detect the levels of assessed miRNAs, including miR -1, -21, -23, and -423-5-p. Also, baseline demographic and clinical characteristics associated with the level of miRNAs were recorded. In the second phase, patients were followed for one year after admission, and several outcomes, including in-hospital mortality, one-year mortality, number of readmissions, and Functional Classification of New York Heart Association (FC) after one year, were recorded (de novo AHF according to Framingham-based criteria following the confirmation of two attending cardiologists were included. Patients with HF with reduced ejection fraction (HFrEF: at least one major Framingham-based criteria/two minor criteria and ejection fraction [EF] <40%) and those with HF with preserved ejection fraction (HFpEF: at least one major criteria/two minor criteria and EF ≥50%) were included. As per established HF guidelines, heart failure with mid-range EF (HFmrEF) was defined as HF with an EF of 40–49%, and those patients with HFmrEF were excluded from our study Reviewers' comments:Reviewer's Responses to Questions  Comments to the Author1. Is the manuscript technically sound, and do the data support the conclusions?  The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: YesReviewer #2: No**********  2. Has the statistical analysis been performed appropriately and rigorously?   Reviewer #1: I Don't KnowReviewer #2: No**********  3. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.  The Reviewer #1: YesReviewer #2: No**********  4. Is the manuscript presented in an intelligible fashion and written in standard English?  PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: NoReviewer #2: Yes**********  5. Review Comments to the Author  Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: May I congratulate the authors on conducting this original review. The reviewed manuscript fits with the Journal's scope, with a moderate clinical importance in the cardiology area and adds value to its field. In the review article entitled "Novel Diagnostic Potential of miR-1 in Patients with Acute Heart Failure", the authors try to assess the prognostic and diagnostic role of some miRNA in acute heart failure patients. The article summarises the risk factors and comorbidities of HFpEF and HFrEF patients and compares the plasma values for miR -1, -21, -23, and -423-5-p in acute heart failure patients and healthy subjects. The patients were followed for one year, including in-hospital mortality, one-year mortality, and the number of readmissions and the measures performed seem to have a diagnostic value.The title is informative and relevant and the research/review question is clearly outlined, however, the authors should consider introducing HFmrEF in the title and in the text as well.The background is clear and tries to shape what is already known about this topic, however it should offer a clearer view why did the authors only assessed this particular types of miRNA: -1, -21, -23, and -423-5-p for acute heart failure patients.The main section needs an appropriate reporting: the data could be presented in a better way (data from the tables and figures could be more clearly presented).Titles, columns and rows are labelled correctly and clearly, however, categories could be grouped more appropriately. The text is repetitive of the data included in tables, although the authors do not convince the reader what is a statistically significant result.The conclusions section answers the aims of the study although it should be better supported by results. The manuscript should state its impact on current and future practice and also its limitations .The references used are relevant, not so recent, referenced correctly and the authors tried to include appropriate key studies, although missed some.Overall, the review was appropriate to answer the aim, based also on previous studies, however, the authors should address the following issues/ flaws of this article:1. All acronyms and abbreviations must be explained in full at first mention . All abbreviations used must be defined and spelt out in full in the caption.2. Author(s) should be consistent when reporting abbreviation throughout the manuscript (eg: HfrEF (line 82) vs HFrEF ; or HfpEF (line 83) vs HFpEF 3. In the section "prevalence and incidence of HFpEF", in paragraph 3, starting with line 50, the reader could get confused as the authors start comparing HFpEF with HFrEF4. In the section entitled "Risk factors and comorbidities of HFpEF", the authors should try and restate the ESC guidelines/ first sentence could be reworded.5. Although the review should refer to HFpEF the authors compare HFpEF with HFrEF 6. Authors should clarify why patients with HFmrEF= heart failure with mildly reduced ejection fraction where excluded from the study – line 84-85 .7. In study limitation, authors mention that most of the patients included in their study had HFpEF, however, as per the result section, most of them had HFrEF.8. Authors should state the limitation more clearly (e.g.what influences the assay/ the test?)Language:1. "cronray" should read as "coronary" (line 63)?2. “freeze-thawing” should read as “defrost/unfreeze”? (line 90)3. A sentence should end with dot “.”(line 90)4. A sentence always starts with capital letter. Authors should reconsider/ re-arrange first sentence in Discussions section. (line208). Also line 235.5. Authors should reconsider second sentence in the conclusion section, line 266: “Therefore, they the miRNAs in particular miR-1 can be taken into account as diagnostic aids for AHF.”Tables and figures:1. For tables and figures, all the abbreviation used should be defined in a footnote (e.g. FC)2. Any Table (Table 2) that require more than 2 pages, must be supplied as supplementary material.3. When reporting in table 1 BMI that should be reported as 23.55 ±6.144. In the healthy control group all the subjects were non-smokers?5. In table 1, when referred to Type of heart failure (based on history), n (%) - “acute on chronic” should read as” acute or chronic”?6. Table 2 needs a better discussion in the text.7. Authors should consider performing a statistical analysis plan and should consider using cluster approach.Reference:1. The first reference used is the guidelines, however it is advisable to use an updated version (current one dates from 2021)2. The references used could be updated and more recent.Reviewer #2: The authors submitted a research article in which they evaluated the potential of selected miRNAs, including miR-1, -21, -23, and -423-5-p, in the diagnosis and prognosis of AHF patients. They randomly selected 85 AHF patients and 58 healthy control individuals. The authors evaluated a signature of miRNAs  and did not find any association between the level of miRNAs prognostic outcomes, whereas diagnostic power of evaluatedmiRNAs  for acute HF were determed. Although these findings are impressive, I would like to put forwrd several issues to comments.1. Methodology: the authors should clearly report what HF was evaluated: chronic HF after recovery of acute HF or acute de novo HF / acutely decompensated HF. Endpoints are needed to be explained. Please, check and add more information. Flow chart with clear inclusion / non-inclusion criteria a re needed.2. Statistics: sample size is required to be calculated. Please, add pertnent formula and an example of estimation. Yet, the validation method is needed to be reported along with comparissions of the models with AUCs, IDI and INR.3. Results: Table 1 does not corresponds to the initial hypothesis of acute HF. The authors should re-check the data and improve them.4. Please, use comparisson of the model with standard model that is required to be used in accordance to current ESC guideline5. The multivariate log regression is needed to evaluate whether additional variables exist and influence prognosis.**********  6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). 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Please note that Supporting Information files do not need this step.While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool,  13 Aug 2022Dr. Emily ChenetteEditor-in-ChiefPLOS ONEDear Dr. Chenette,Subject: Revised Manuscript, Authors' Responses Thank you for your email and favorable response and comments. We appreciate the reviewers’ constructive suggestions. Please find attached the revised manuscript. Major revisions have been made to improve the manuscript, as suggested. The revisions and new citations have improved the content. Reformatting and editing also made a seamless flow of information.The changes are marked by Track Changes. Below are the authors’ responses to the comments of the reviewers.We hope that these revisions are acceptable and the manuscript can go forward for publication in PLOS ONE.Thanks and Regards,Reza Badalzadeh, PhDCardiovascular Research centerMadani Hospital, Tabriz University of Medical SciencesTabriz, Iran Tel: +984133373919Fax: +984133373910reza.badalzadeh@gmail.comEmail: Authors' Responses are colored in blueEditor Comments:Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.Specifically, check the statistics performed and adjust with proper methods. Report the event-per variable ration and the strategy to avoid overfitting. Please report the comparison for diagnostic accuracy in the ROC curves. The event-per-variable ratios for those with multivariable binary regression analysis were added to the footnote of Table 3. A statement regarding this issue was added to the limitation of the study section. Although it is believed that the evidence underlying the rule of EPV = 10 as a minimal sample size criterion for binary logistic regression analysis is weak [1].Comparison for diagnostic accuracy in the ROC curves of each miRNA was added as a new column to Table 2.Comment:1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdfThe manuscript format was changed to meet the style of PLOS ONEComment:2. Please amend your current ethics statement to address the following concerns:a) Did participants provide their written or verbal informed consent to participate in this study?b) If consent was verbal, please explain i) why written consent was not obtained, ii) how you documented participant consent, and iii) whether the ethics committees/IRB approved this consent procedure.“Written informed consent was obtained from all participants.” This statement was added to the ethics statement section.Comment:http://journals.plos.org/plosone/s/data-availability.3. In your Data Availability statement, you have not specified where the minimal data set underlying the results described in your manuscript can be found. PLOS defines a study's minimal data set as the underlying data used to reach the conclusions drawn in the manuscript and any additional data required to replicate the reported study findings in their entirety. All PLOS journals require that the minimal data set be made fully available. For more information about our data policy, please see https://data.4tu.nl/info/en/use/publish-cite/upload-your-data-in-our-data-repository) Regarding the “Data Availability statement”, we added a reasonable phrase fit into this query, in the end of the manuscript before references, if it is not acceptable we can also prepare the minimal data set as supplementary files, if not, the required data could be uploaded in a standard repository system for public access. , and click on the Fetch/Validate link next to the ORCID field. This will take you to the ORCID site and allow you to create a new iD or authenticate a pre-existing iD in Editorial Manager. Please see the following video for instructions on linking an ORCID iD to your Editorial Manager account: The ORCID iD was added.Comment:5. Please ensure that you refer to Figure 1 in your text as, if accepted, production will need this reference to link the reader to the figure.Figure 1 was referred to in the results section, paragraph 3.Additional Editor Comments:Please clarify what the authors mean for ICD Implementation. Is it Implantation or upgrade? Also clarify the timing of ICD implantation and, when correcting, using it as a time dependent variable or removing it from the outcomes.The ICD implementation was removed from the outcomes.Comment:Please report the HFA-PEFF and the HF2PEF scores before classifying HFpEF.Heart failure with preserved ejection fraction was defined based on left ventricular ejection fraction in this study. Considering that E/e′ ratio was not measured for our patients we are unable to report HFA-PEFF and the HF2PEF scores.REVIEWER 1Comment:May I congratulate the authors on conducting this original review. The reviewed manuscript fits with the Journal's scope, with a moderate clinical importance in the cardiology area and adds value to its field. In the review article entitled "Novel Diagnostic Potential of miR-1 in Patients with Acute Heart Failure", the authors try to assess the prognostic and diagnostic role of some miRNA in acute heart failure patients. The article summarises the risk factors and comorbidities of HFpEF and HFrEF patients and compares the plasma values for miR -1, -21, -23, and -423-5-p in acute heart failure patients and healthy subjects. The patients were followed for one year, including in-hospital mortality, one-year mortality, and the number of readmissions and the measures performed seem to have a diagnostic value.The title is informative and relevant and the research/review question is clearly outlined, however, the authors should consider introducing HFmrEF in the title and in the text as well.We highly appreciate your comments and positive feedback. HFmrEF definition was added to the methods section and related reference was cited.Comment:The background is clear and tries to shape what is already known about this topic, however it should offer a clearer view why did the authors only assessed this particular types of miRNA: -1, -21, -23, and -423-5-p for acute heart failure patients.A thorough explanation of the reasons for the selection of these miRNAs was added to the introduction section. These miRNAs were mainly selected to include the most representative miRNAs that play different roles in cardiovascular pathology: hypertrophy, inflammation, fibrosis, acute coronary syndrome, arrhythmia:MiR-1 is one of the most abundant miRNAs in cardiac tissue and is linked to cardiac remodeling being inversely correlated with hypertrophy. Experimental animal studies showed that adenoviral delivery of miR-1 was able to reverse hypertrophy . It plays also a role in atrial and ventricular arrhythmias. For example, it has been shown that its levels are markedly reduced in patients with atrial fibrillation. It also plays a role in acute coronary syndromes, a hypothesis that has yet to be further validated [4].MiR-21 plays a role in inflammation and smooth muscle proliferation and fibrosis. It has been found to be increased in mice hearts subjected to ischemia-reperfusion and more specifically in fibroblasts within the infarct zone . It plays also a profibrotic role in heart failure from causes other than ischemia (i.e. aortic stenosis) through induction of matrix metalloprotease-2 (MMP-2) production. On the other hand, it attenuates cardiomyocyte apoptosis secondary to oxidative stress [3].MiR-23 is upregulated in cardiac hypertrophy and promotes hypertrophy, while antagomiR-mediated silencing of miR-23a reverses it . Finally, miR-423-5-p has been also found to be increased in HF. In a small study, Tijsen et al. showed that miR-423-5-p strongly discriminated HF-associated dyspnea from healthy controls (C-statistic 0.91) and non-HF dyspnea (C-statistic 0.83) and was correlated with NT-proBNP and LVEF [16]. In another study comparing several miR levels in patients with dilated cardiomyopathy and age- and sex-matched healthy controls, miR-423-5-p was found to have modest discrimination power (C-statistic 0.67) and was also correlated with NT-proBNP levels [5]. A 2020 systematic review showed that miR-423-5-p was among 5 miRNAs that were differentially expressed in more than one included studies [6]. Furthermore, miR-423-5-p levels in plasma may be time-dependent. A study of 246 post-MI patients showed that miR-423-5-p levels rose over time from index hospitalization to 3 months and up to 1 year post-discharge [7].On the role of miRNAs in HF prognosis, a very recent systematic review and meta-analysis showed that HF patients with low expression of miR-1, miR-21, miR-23, and miR-423-5-p have significantly worse overall survival. However, among them, miR-423-5-p is the stronger biomarker for HF prognosis [8].Many experimental animal, preclinical, and clinical human studies and reviews have tried to explore the role of miR in HF diagnosis with much attention focused on their relative superiority or non-inferiority compared to established biomarkers such as BNP or NT-proBNP . MicroRNAs may predict the development of HF at an earlier stage as they play a pathophysiological role in myocardial hypertrophy, fibrosis and apoptosis prior to overt clinical symptoms. Despite the positive results of some studies, the clinical utility of these miRNAs in clinical practice is yet to be determined. Here, we aimed to evaluate the potential of selected miRNAs, including miR-1, -21, -23, and -423-5-p, in the diagnosis and prognosis of AHF patients.Comment:The main section needs an appropriate reporting: the data could be presented in a better way (data from the tables and figures could be more clearly presented).The design of the tables was improved to make them more clear. Nevertheless, upon you find it inadequate or have any specific suggestions please kindly guide us with more detailed explanation. Comment:Titles, columns and rows are labelled correctly and clearly, however, categories could be grouped more appropriately. The text is repetitive of the data included in tables, although the authors do not convince the reader what is a statistically significant result.The data in tables were grouped to show the results more clearly. The results section was improved accordingly.Comment:The conclusions section answers the aims of the study although it should be better supported by results. The manuscript should state its impact on current and future practice and also its limitations .The conclusion was edited and the impact of the findings of our study was added. The limitations of measurement and detection of miRNAs as added to the study limitation section.Comment:The references used are relevant, not so recent, referenced correctly and the authors tried to include appropriate key studies, although missed some.The references were updated accordingly.Comment:Overall, the review was appropriate to answer the aim, based also on previous studies, however, the authors should address the following issues/ flaws of this article:1. All acronyms and abbreviations must be explained in full at first mention . All abbreviations used must be defined and spelt out in full in the caption.The abbreviations and acronyms were defined in the text and also the caption of Tabels.Comment:2. Author(s) should be consistent when reporting abbreviation throughout the manuscript (eg: HfrEF (line 82) vs HFrEF ; or HfpEF (line 83) vs HFpEF The consistency of abbreviation was checked and the mentioned mistakes were corredcted..Comment:3. In the section "prevalence and incidence of HFpEF", in paragraph 3, starting with line 50, the reader could get confused as the authors start comparing HFpEF with HFrEFWe could not find this section in our manuscript. Would you please check again the section? It may have been already corrected or it could be related to another manuscript.Comment:4. In the section entitled "Risk factors and comorbidities of HFpEF", the authors should try and restate the ESC guidelines/ first sentence could be reworded.We could not find this section in our manuscript. Would you please check again the section? It may be related to another manuscript.Comment:5. Although the review should refer to HFpEF the authors compare HFpEF with HFrEF We could not find the mentioned issue in the listed lines in our manuscript. Would you please check again the section?Comment:6. Authors should clarify why patients with HFmrEF= heart failure with mildly reduced ejection fraction where excluded from the study – line 84-85 .An explanation was added to the methods section.Comment:7. In study limitation, authors mention that most of the patients included in their study had HFpEF, however, as per the result section, most of them had HFrEF.The sentence was edited.Comment:8. Authors should state the limitation more clearly (e.g.what influences the assay/ the test?)The limitations section was completed.Comment:Language:1. "cronray" should read as "coronary" (line 63)?2. “freeze-thawing” should read as “defrost/unfreeze”? (line 90)3. A sentence should end with dot “.”(line 90)4. A sentence always starts with capital letter. Authors should reconsider/ re-arrange first sentence in Discussions section. (line208). Also line 235.5. Authors should reconsider second sentence in the conclusion section, line 266: “Therefore, they the miRNAs in particular miR-1 can be taken into account as diagnostic aids for AHF.”The language mistakes were corrected.Comment:Tables and figures:1. For tables and figures, all the abbreviation used should be defined in a footnote (e.g. FC)The abbreviations were accordingly completed.Comment:2. Any Table (Table 2) that require more than 2 pages, must be supplied as supplementary material.Tables were edited to fit on one page. Thank you.Comment:3. When reporting in table 1 BMI that should be reported as 23.55 ±6.14The style of reporting, “the mean ± SD” was corrected.Comment:3. In the healthy control group all the subjects were non-smokers?Thank you for nice query. The prevalence of smoking among healthy subjects was added.Comment:4. In table 1, when referred to Type of heart failure (based on history), n (%) - “acute on chronic” should read as” acute or chronic”?“Acute on chronic heart failure” was named according to the ICD-10-CM Code: I50. 23 as an acute heart failure occured following the chronic heart failure”.Comment:5. Table 2 needs a better discussion in the text.The discussion over Table 2 was added to the discussion section. Comment:6. Authors should consider performing a statistical analysis plan and should consider using cluster approach.A flowchart of the statistical analysis plan and approach was added as supplementary figure 2.Comment:Reference:1. The first reference used is the guidelines, however it is advisable to use an updated version (current one dates from 2021)2. The references used could be updated and more recent.The reference list was updated.REVIEWER 2The authors submitted a research article in which they evaluated the potential of selected miRNAs, including miR-1, -21, -23, and -423-5-p, in the diagnosis and prognosis of AHF patients. They randomly selected 85 AHF patients and 58 healthy control individuals. The authors evaluated a signature of miRNAs  and did not find any association between the level of miRNAs prognostic outcomes, whereas diagnostic power of evaluated miRNAs  for acute HF were determed. Although these findings are impressive, I would like to put forwrd several issues to comments.Comment:1. Methodology: the authors should clearly report what HF was evaluated: chronic HF after recovery of acute HF or acute de novo HF / acutely decompensated HF. Endpoints are needed to be explained. Please, check and add more information. Flow chart with clear inclusion / non-inclusion criteria are needed.We included patients with acute decompensation of CHF  heart failure) or de novo acute heart failure. This explanation was added to the methods section.Comment:2. Statistics: sample size is required to be calculated. Please, add pertnent formula and an example of estimation. Yet, the validation method is needed to be reported along with comparissions of the models with AUCs, IDI and INR.The sample size calculation method was added. Validation models including AUC and INR were also added to the supplementary table.Comment:3. Results: Table 1 does not corresponds to the initial hypothesis of acute HF. The authors should re-check the data and improve them.The data in Table was re-checked and corrected.Comment:4. Please, use comparisson of the model with standard model that is required to be used in accordance to current ESC guidelineThe AUC for each miRNA is reported for comparison with standard model.Comment:5. The multivariate log regression is needed to evaluate whether additional variables exist and influence prognosis.The multivariate log regression has been already performed but due to the low number of outcomes and the proability of overfitting, we were not able to add more variables to our model. This issue was adderessed in our study limitations section.References1. van Smeden M, de Groot JAH, Moons KGM, Collins GS, Altman DG, Eijkemans MJC, et al. No rationale for 1 variable per 10 events criterion for binary logistic regression analysis. BMC Med Res Methodol. 2016;16(1):163. doi: 10.1186/s12874-016-0267-3.2. Melman YF, Shah R, Das S. MicroRNAs in heart failure: is the picture becoming less miRky? Circ Heart Fail. 2014;7(1):203-14.https://doi.org/10.1016/j.bbadis.2016.11.034.3. Wang H, Cai J. The role of microRNAs in heart failure. Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease. 2017;1863(8):2019-30. doi: 4. Romaine SPR, Tomaszewski M, Condorelli G, Samani NJ. MicroRNAs in cardiovascular disease: an introduction for clinicians. Heart. 2015;101(12):921-8.5. Fan K-L, Zhang H-F, Shen J, Zhang Q, Li X-L. Circulating microRNAs levels in Chinese heart failure patients caused by dilated cardiomyopathy. Indian Heart J. 2013;65(1):12-6.6. Peterlin A, Počivavšek K, Petrovič D, Peterlin B. The role of microRNAs in heart failure: a systematic review. Frontiers in Cardiovascular Medicine. 2020;7:161.7. Bauters C, Kumarswamy R, Holzmann A, Bretthauer J, Anker SD, Pinet F, et al. Circulating miR-133a and miR-423-5p fail as biomarkers for left ventricular remodeling after myocardial infarction. Int J Cardiol. 2013;168(3):1837-40.8. Yang J, Yang X-S, Fan S-W, Zhao X-Y, Li C, Zhao Z-Y, et al. Prognostic value of microRNAs in heart failure: A meta-analysis. Medicine. 2021;100(46).Attachmentresponse to reviewers 5.docxSubmitted filename: Click here for additional data file. 9 Sep 2022Novel Diagnostic Potential of miR-1 in Patients with Acute Heart FailurePONE-D-22-15097R1Dear Dr. Badalzadeh,We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at onepress@plos.org.If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact Kind regards,Prof. Raffaele Serra, M.D., Ph.DAcademic EditorPLOS ONEAdditional Editor Comments :amended manuscript is acceptableReviewers' comments:Reviewer's Responses to Questions  Comments to the Author  1. 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The multivariance analysis revealed a significant relationship between survival and TTF-1 (p = 0.034) and ECOG score (p = 0.007). We found a correlation between survival time and ECOG performance score and TTF-1. TTF-1 can be used as a biomarker to monitor prognosis in the follow-up and treatment of patients with AC that develop BM.Brain metastases (BMs) are common in lung adenocarcinomas (ACs). Thyroid transcription factor 1 (TTF-1) is important in the diagnosis of AC. This study aimed to examine the relationship between TTF-1 and BM for the first time in literature. The data of 137 patients with AC that developed BM between 2009 and 2020 were retrospectively analyzed. A total of 137 patients, 120 (87.6%) male, and 17 (12.4%) female were examined. Their mean age was 59.78 ± 0.82 years. The Eastern Cooperative Oncology Group (ECOG) performance score was 0–1 (< 2) for 39 (28.5%) patients and 2–4 (≤ 2) for 98 (71.5%). TTF-1 was positive in 100 (73%) patients and negative in 37 (27%). More than five BMs were present in 102 (74.4%) patients and less than five in 35 (25.6%). All the patients received whole-brain radiotherapy. None of the patients was suitable for surgery or radiosurgery. The median survival time was 6.4  months. The survival time was 7  months for the TTF-1 (+) patients and 5.8  months for the TTF-1 (−) patients. In the univariate analysis, there was a significant relationship between survival time and age ( Lung cancer constitutes 40–60% of BM4. The most common non-small cell lung cancer that metastasizes to the brain is lung adenocarcinoma (AC). Thyroid transcription factor 1 (TTF-1) is a transcription protein that is positive in 70–80% of ACs, and it is used in the diagnosis of AC5. In recent years, studies have shown that TTF-1 is not only an important diagnostic but also a prognostic and predictive marker in patients with Acs6. However, the relationship between BM in AC and TTF-1 remains unknown. Therefore, in this study, we retrospectively evaluated AC cases that developed BM during their follow-up in our center.Due to the advances in diagnosis and treatment in recent years, the life expectancy of cancer patients has been prolonged. However, there has been no decrease in the incidence of brain metastases (BM)Patients who were followed up with a diagnosis of AC and developed BM between 2009 and 2020 in the Medical Oncology Department of Manisa City Hospital were retrospectively analyzed. The study included patients with stage 4 AC, aged 18 years or older, who were found to have BM at the time of diagnosis or developed BM during their follow-up. The patients were negative for c-ros oncogene 1(ROS-1), anaplastic lymphoma kinase (ALK), and epidermal growth factor receptor (EGFR) mutation. The BM diagnosis was made based on brain magnetic resonance images.The patient's demographic characteristics, such as age and sex, ECOG performance scores, number of BM, TTF-1 results, and their relationship with survival were examined. TTF-1 was examined from the first lung biopsies of the patients at the time of diagnosis in all patients Sections of 3–5 micron thickness were prepared for the immunohistochemical examination. Ventana brand fully automatic staining device  was used for immunohistochemical studies. TTF-1  as primary antibody was manually instilled and incubated at 37 °C. Pneumocytes were evaluated as an internal control for TTF-1, and nuclear staining was considered positive. The patients were divided into groups according to the number of BM (< 5 and ≥ 5), the Eastern Cooperative Oncology Group (ECOG) performance score (< 2 and ≥ 2), and age . To obtain the survival time of the patients, the time from the date of diagnosis to mortality or the time to the last follow-up for the patients who survived was calculated.t-test in case of normal distribution and the Mann–Whitney U test otherwise. Rates were compared between the groups using the chi-square analysis and Fisher’s exact test. Survival analyses were undertaken with the Kaplan–Meier method. Determinative factors were examined using the Cox regression analysis. p < 0.05 was considered as significant in all statistical analyses.Descriptive statistics were presented as mean, standard deviation, median, minimum and maximum values for numerical variables, and as numbers and percentages for categorical variables. The comparison of numerical variables between two independent groups was performed using Student’s The study was conducted by the principles of the Declaration of Helsinki and reviewed and approved by the Health Sciences Ethics Committee of Manisa Celal Bayar University . This study was designed retrospectively. Informed consent has been waived by the Health Sciences Ethics Committee of Manisa Celal Bayar University.p = 0.047), TTF-1 (p = 0.024), and ECOG performance score (p = 0.002)  and ECOG performance score (p = 0.007)  male, and 17 (12.4%) female were examined. Their mean age was 59.78 ± 0.82 years. Thirty-nine (28.5%) patients had an ECOG performance score of 0–1 while 98 (71.5%) had an ECOG performance score of 2–4. TTF-1 was positive in 100 (73%) patients and negative in 37 (27%). More than five BM were present in 102 (74.4%) patients and less than five in 35 (25.6%)  patients was longer, and this was statistically significant in the multivariance analysis independent of the number of BM, ECOG performance score, and age. This study is important since there is no previous research examining this relationship.23. These results have also been supported by meta-analyses26. TTF-1-positive lung cancers have a better prognosis. It is unknown why TTF-1-positive patients have a better prognosis. However, Myong et al. showed that TTF-1 expression was inversely proportional to the Ki-67 proliferation index27. The inverse correlation of Ki-67 with TTF expression may be one reason for the difference between TTF-1 positive and TTF-1 negatives. Although all of the patients in our study were negative for EGFR mutation, it has been reported in the literature that higher rate of EGFR mutations were detected in TTF-1 positive patients than in TTF-1 negative patients29. Patients with EGFR mutations have a longer life expectancy with the use of tyrosine kinase inhibitor drugs32. This may be another reason for the difference between TTF-1 positives and TTF-1 negatives. On the other hand, the respiratory tract is divided into the terminal respiratory unit (TRU), which plays a role in oxygen-carbon dioxide exchange, and the non-TRU, which plays a role in conducting air intake. TRU contains TTF-1-expressing cells, while non-TRU contains non-TTF-1-expressing cells33. TTF-1-positive and TTF-1-negative tumors may develop from different parts of the respiratory tract and may show different clinical courses. Another reason for the difference between TTF-1 positive and TTF-1 negative tumors may be that TTF-1 positive and negative tumors developed from different regions of the respiratory tract.Studies examining the relationship between AC and TTF-1 have reported that TTF-1 (+) patients have a better response to chemotherapy and have longer life expectancy in both early and advanced stagesIn our study, TTF-1 was evaluated from the first lung biopsies at the time of diagnosis. TTF-1 may differ between primary tumors and metastasis. But Brain metastasis is not biopsied in lung cancers because radiological findings are often sufficient in the diagnosis of brain metastases and the clinical conditions of the patients are not suitable for cranial biopsy in daily practice. This is one of the weaknesses of this study. In addition, grading data could not be obtained In this study. Because all of our cases had advanced diseases. For this reason, they were diagnosed with biopsies instead of surgical applications. Materials taken for diagnostic biopsies are usually small, so grading and differentiation are not always appropriate. Since our study was retrospective, a new biopsy could not be taken again. This is another weak point of the study.Examination of TTF-1 only in lung biopsies in the study, and the retrospective, single-centered study with a small number of patients are the limitations of our study. However, our study is important because it is the first study to show the relationship between TTF-1 and BM.In conclusion, TTF-1 presents as an easily obtained, inexpensive, and useful biomarker to determine prognosis in patients with AC that develop BM, when used together with the ECOG performance score.


Significantly, pro-apoptotic tumor necrosis factor (TNF) peptide (P13) was found to inhibit HIV-related cell death and reduced viral transmission. Whereas pro-necrotic TNF peptide (P16) had little effect on HIV-related cell death and viral transmission. Understanding mechanisms by which cell death can be manipulated may provide additional drug targets to reduce the loss of CD4+ cells and the formation of a viral reservoir in HIV infection.The human immunodeficiency virus (HIV) is still a global pandemic and despite the successful use of anti-retroviral therapy, a well-established cure remains to be identified. Viral modulation of cell death has a significant role in HIV pathogenesis. Here we sought to understand the major mechanisms of HIV-induced death of lymphocytes and the effects on viral transmission. Flow cytometry analysis of lymphocytes from five latent HIV-infected patients, and HIV IIIB-infected MT2 cells demonstrated both necrosis and apoptosis to be the major mechanisms of cell death in CD4 Further understanding of HIV-mediated cell death may lead to the development of further therapeutic targets.Apoptosis is a type of programmed cell death whereby cells initiate their own demise in response to a stimulus, which has been a well-studied mechanism of cell death in HIV infection , 4. Casppatterns . Necrosid tissue . Other cd tissue , 17. Thed tissue . DespiteTargeting cell death as a treatment for HIV is an attractive idea , 19. HowThe aim of this study is to identify cell death patterns in patients with latent HIV infection (anti-HIV antigen positive without any HIV-related symptoms) and to understand the consequences of apoptosis and necrosis on HIV-infected cells. The key reagents used in this project are two peptides derived from tumor necrosis factor (TNF). TNF binds to the tumor necrosis factor receptors (TNFR) and causes pro-inflammatory signaling or cell death . TNF mayThe goal of this study was to understand the type of cell death in latent HIV infection and the effects of two different cell deaths, apoptosis or necrosis, on viral transmission. We first recruited five patients newly diagnosed with HIV infection through an HIV clinic in China. The cohort was tested for HIV at regular screenings and diagnosed using western blots. These patients did not have any HIV-related symptoms when they were recruited in this study. Healthy volunteer adults between the ages of 20 and 45 were eligible for enrollment. We conducted the studies in compliance with Good Clinical Practice guidelines (CPMP/ICH/135/95) and the Declaration of Helsinki. They were all studied with informed consent in full compliance with national and institutional ethical requirements . All samples were stored at −80°C until analysis.2. Human TNF was purchased from Immunotools . Peptides were synthesized on an automatic APEX 396 using a standard solid phase Fmoc strategy. In some of the validation experiments, peptides (e.g. P13 and P16 peptides) were purchased from Proimmune . HIV-1 strain IIIB (1000 TCID50) kindly provided by Professor Tao Dong  was used to infect cells in vitro.MT2 cells were purchased from American Type Culture Collection  and maintained in RPMI supplemented with 10% fetal calf serum and penicillin/streptomycin (complete medium). All cultures were incubated at 37°C in 5% COHIV-1 IIIB virus at a multiplicity of infection of 0.001 was adsorbed to MT2 cells in serum-free RPMI, 1 mM L-glutamine, 0.1% bovine serum albumin for 1 hour at 37°C. Cells were then supplemented with the complete medium in the presence or absence of DMSO-dissolved whole TNF protein or TNF-derived peptides and incubated for additional 24 hours at 37°C. The cells were stained with live/dead cell-staining kits  according to the manufacturer’s instructions and fixed using cytofix/cytoperm fixation/permeabilisation solution kits . This was followed by intracellular staining with the FITC-conjugated anti-caspase-3 antibody . HIV-1 Gag p24 protein was detected by PE-conjugated anti-p24 monoclonal antibody . All other flow cytometry antibodies were purchased from BD Biosceinces. Caspase inhibitor z-VAD  was dissolved in DMSO and used at 10 µM in this study. Cells were acquired on a CyAn flow cytometer  and data were analyzed using Flowjo .p < 0.05, ** p < 0.01, *** p < 0.001, and ns .Comparisons for two groups were calculated by multiple, unpaired two-tailed t-tests with Prism 9 . Statistical significance was defined at the following levels: * via its receptor, CD4, together with its co-receptor, CCR5 or CXCR4, ultimately causing cell death (via flow cytometry. A higher degree of cell death by apoptosis (Caspase-3+ live-dead−) and necrosis (Caspase-3− live-dead+) was found in both CD4+ CD8- T cells and double negative  lymphocytes of the patients than in CD4- CD8+ T cells and those of the healthy individuals , or DMSO (no peptide control) for 24 hours. Induction of cell death was identified by caspase-3 and live-dead dye double staining and measured by flow cytometry. Differential peptide-induced cell death was found at concentration of 100 μM, but not so distinct at 6 or 25 μM. At the baseline level, ~5% of cell death from apoptosis and ~18% from necrosis. Addition of P13 at 100 μM induced a particular high frequency (28%) of apoptotic (Caspase-3+ live-dead−) (− live-dead+), this was in agreement with our previous finding (We have previously identified peptide fragments within TNF that are responsible for its pro-apoptotic and pro-necrotic functions . We firs− live-dead− cells) decreased significantly less rapidly in HIV-infected cells incubated with P13 over 4 days in comparison to infected cells without exposure of the peptide  in HIV-infected MT2 cells compared with non-infected cells. Viral load increased in live, necrotic and apoptotic MT2 cells over 4 days of HIV infection  lymphocytes, likely T, B, and natural killer (NK) cells, died by apoptosis and necrosis. As they are not targeted by the virus like CD4+ and CD8+ T cells, suggesting the presence of a bystander effect. One possible explanation for this bystander effect in HIV patients is that the virus can infect and kill CD4+ and CD8+ T cells, which can release signaling molecules, such as cytokines or chemokines, that can induce necrosis in neighboring cells, including double-negative lymphocytes. Additionally, the immune response mounted against the virus can also contribute to the destruction of neighboring cells by the release of cytotoxic molecules from the activated immune cells. The death of these double negative lymphocytes may contribute to the impairment of the immune system. Studies have also found that HIV infection can cause the loss of regulatory T (Treg) cells  cells  and thatg) cells  might exeservoir . Furthereservoir , 31. FurIn this study, we sought to identify the major types of cell death in HIV infection and the effects of TNF peptides on latent infection. The finding of this study suggests that induction of apoptosis in HIV infected cells benefits in restriction of HIV spreading. Previously, models of HIV activation by inducing apoptosis have been tested in order to target the latent reservoir . FurtherThe original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding authors.The studies involving human participants were reviewed and approved by Ethics Committee, Beijing Youan Hospital, Capital University, Beijing, China. The patients/participants provided their written informed consent to participate in this study.QC performed the majority of experiments and data analysis. WL and C-HC conducted some of the experiments and analysis. YHZ, YZ and JZ collected and provided clinical samples. C-HC and SJ designed the project. QC, C-HC, and SJ wrote the manuscript. All authors contributed to the article and approved the submitted version.


Schizophrenia is a severe psychiatric disorder with a high prevalence worldwide, however, its pathogenesis remains poorly understood.In this study, we used the non-competitive NMDA receptor antagonist MK-801 to induce schizophrenia-like behaviors and confirmed that mice exhibited stereotypic rotational behavior and hyperlocomotion, social interaction defects and cognitive dysfunction, similar to the clinical symptoms in patients. Here, the anterior cingulate cortex (ACC) and basolateral amygdala (BLA) were involved in the schizophrenia-like behaviors induced by MK-801. Furthermore, we confirmed BLA sent glutamatergic projection to the ACC. Chemogenetic and optogenetic regulation of BLA-ACC projecting neurons affected social and cognitive deficits but not stereotypic rotational behavior in MK-801-treated mice.Overall, our study revealed that the BLA-ACC circuit plays a major role and may be a potential target for treating schizophrenia-related symptoms. Schizophrenia is a chronic, severe psychiatric disorder with a global prevalence of approximately 1% . ClinicaIn recent years, researchers have shown an increased interest in the abnormalities of glutamatergic neurotransmission in individuals with schizophrenia . The redDespite the aforementioned discoveries, the understanding of the pathology of schizophrenia is far from sufficient. The pathogenesis of schizophrenia is notably very complex. It is characterized by multiple clinical symptoms, indicating that far more than one brain region is involved. Previous studies have shown that surgical ablations of certain corticolimbic regions, such as the anterior cingulate cortex (ACC), hippocampal formation (HIP), and basolateral amygdala (BLA) , result In the current study, we used a mouse model of MK-801-induced schizophrenia to determine the roles of the ACC and BLA in schizophrenia-like behaviors. Furthermore, we evaluated the effect of modulating the neural circuitry on these behaviors to understand its etiology and increase our knowledge of the brain pathology in individuals with schizophrenia.ad libitum. All experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC-20200356) of the Fourth Military Medical University and were performed according to the “Principles of Medical Laboratory Animal Care” issued by the National Ministry of Health in China. All efforts were made to minimize animal suffering and to reduce the animals number used in this study.We obtained adult C57BL/6J mice from the Fourth Military Medical University (FMMU) animal facility. Glutamic acid decarboxylase-green fluorescence protein (GAD67 GFP) mice were a kind gift from Dr. Nobuaki Tamamaki, Kumamoto University . GCaMP6sAdult male C57BL/6 mice (6–8 weeks) were obtained from the Experimental Animal Center of Fourth Military Medical University  and then were weighed and randomly assigned to the control group or the experimental group at a 1:1 ratio. The mice were acclimated for at least 7 days in the holding room before the experimental test, and they were gently handled for 3–5 min each day by the experimenters for seven consecutive days before behavior tests. For optogenetic manipulations and fiber photometry recording, mice were connected to the patch cable and then allowed to recover for 1–3 min before all behavioral tests were initiated. Considering the possible effects of multiple behavioral tests, different groups were used to explore positive behavior, negative behavior, and cognitive behavior. After the end of the behavioral tests, the brain tissues were removed from the mice for immunofluorescence histochemistry, and the virus injection sites were confirmed, the mice with absence or mis-placed virus injection, incorrect fiber position or drop of ceramic cartridge were excluded from experiments.(+)-MK-801-hydrogen-maleate  was freshly dissolved in Dimethyl sulfoxide (DMSO) first, and then used sterilized normal saline to dilute to the different concentration for intraperitoneal injected. Based on the results of our preliminary experiment, 0.2 mg/kg was finally selected, and the behavioral recording was started 15 min after the injection. The control group of mice was injected with the same volume of vehicle in the same manner. For the Designer Receptor Exclusively Activated by Designer Drugs (DREADD) manipulation before behavioral experiments, DREADD agonist 21 dihydrochloride  was freshly dissolved in 0.9% sterile saline and intraperitoneally administered 3 weeks after DREADD protein expression and 30 min before behavioral tests were performed at a dose of 2 mg/kg.Before the behavioral tests, the mice were gently transferred to the testing room and allowed to acclimate to the environment for at least 1 h. The lighting was ∼ 100 lux in the experimental room to reduce the stimulation of the mice. All apparatuses were cleaned with 75% ethanol at the end of each behavioral test to remove olfactory cues.According to diagnostic and statistical manual of mental disorders 5th ed. (DSM-V) diagnostic criteria, the symptoms of schizophrenia are mainly divided into three categories: positive symptoms, negative symptoms, and cognitive impairment . We compThe rotational behavior of mice was measured in a small non-transparent plastic box (20 cm × 20 cm × 25 cm) after the MK-801 or vehicle injection. An infrared camera mounted on the top of the box was used to record behavior. During the concentration test and incubation period assessment, the behavioral recording started immediately after drug injection until the rotational behavior gradually decreased. Finally, according to the previous experimental results, we chose to conduct recordings 15 min after the drug injection, and each mouse was recorded for 10 min. The experimental device was cleaned thoroughly with 75% alcohol after each trial. The total rotating laps performed by the mouse were analyzed using SMART v.3.0 software  by the experimenters who were blinded to the groups.The OFT was used to evaluate the locomotor ability of mice, as previously described . The OFTA 10-min free social interaction test was performed to examine social behavior when subject animals freely interacted with a juvenile animal, as previously described . The tesThe NOR test was performed as previously described with minor modifications . Mice weCatWalk XT  was used as previously reported to quantitatively assess gait function and motor performance as a complication of schizophrenia . The app–1 using a microsyringe pump . The needle was held at the site for 10 min to allow virus diffusion after injection. For the analysis of the projection of nerve fibers between two brain regions, 150 nL of rAAV-hSyn-EGFP-WPRE-pA  was unilaterally injected into the BLA  of C57BL/6J mice, and 100 nL of rAAV2/retro-hSyn-Cre-mCherry-WPRE-hGH pA virus  was unilaterally injected into the ACC  of GAD67-GFP mice. For behavioral experiments, AAVs were bilaterally injected to the ACC or BLA. For the DREADD activation and subsequent behavioral tests, 150 nL of rAAV-EF1a-DIO-(Gq/GFP/Gi)-WPRE-bGHpA  were bilaterally injected into the BLA, and 100 nL of AAV2/Retro-hSyn-NLS-Cre-P2A-mCherry were bilaterally injected into the ACC. For optical activation and behavioral experiments, 150 nL of rAAV-hSyn-(ChR2/mCherry/NpHR)-WPRE-pA  were bilaterally injected into the BLA, and optical fibers  were implanted in the bilateral ACC  at a 20 degrees-angle on the same day after the viral injection. The coordinates for the optic fiber were 200–300 mm above the viral injection coordinates. For fiber photometry recording, optical fibers  were implanted immediately in the unilateral ACC  and BLA  of the Gcamp6s mice. None of the mice underwent any behavioral tests for at least 3 weeks to allow them to recover from the surgeries. Data from only a post hoc analysis of viral targeting indicated that the injection coordinates were accurate.For the virus injection, C57BL/6J mice were anesthetized with isoflurane, and their heads were fixed in a stereotaxic injection frame . All viral injections were performed using a 10-μL microinjection needle  to deliver the virus at a rate of 30 nL minFor the DREADD manipulation and behavioral experiments, C21 was freshly dissolved in sterile normal saline and intraperitoneally administered 3 weeks after the AAV2/8-EF1a-DIO-hM3D (Gq/GFP/Gi) injection and 30 min before behavioral tests were performed at a dose of 2 mg/kg based on our earlier experimental basis and previous reports .For optical stimulation in the behavioral tests, the patch cord was connected to the laser, and the fiber could freely rotate through a fiber-optic rotary connected with the patch cord. For the rotational behavior, OFT and NOR tests, photoactivation of ACC projecting fiber terminals from BLA was induced by applying 20-Hz light trains with 5-ms pulses of blue light generated by a 473-nm diode-pumped solid-state laser  or constant yellow light generated by a 594-nm diode-pumped solid-state laser  with the light power around 10 mW in the terminal of patch cord. For the social interaction test and OFT, the 10-min test was divided into two 5-min phases: laser stimulation off and on (OFF-ON phases). For the rotational behavior, the 30-min test was divided into three 10-min phases: laser stimulation off, on, and off (OFF-ON-OFF phases).Optical fibers were implanted into the ACC and BLA of Gcamp6s mice. An optical fiber guided the light between the commutator and the implanted optical fiber. The laser power at the tip of the optical fiber was adjusted to 0.01–0.02 mW to decrease laser bleaching. Fluorescence was bandpass-filtered , and an amplifier was used to convert the photomultiplier tube current output to a voltage signal. The analog voltage signals were digitalized at 50 Hz and recorded by a fiber photometry system (Thinker Tech). Fiber photometry system  is equipped with high-speed behavior industrial camera  system. This behavior camera can be controlled by software to output transistor-transistor logic (TTL) signal through general-purpose input/output (GPIO) ports synchronized with each frame and acquired by Data Acquisition Box , which also controlled the fiber photometry acquisition frequency. The data of fiber photometry is acquired by ThinkerTech MultiChannel FiberPhotometry GUI Software based on Labview . In a recording cycle, the number of behavior events divided by the time is the event frequency, which we defined as trail. In each trail, behavior onset has different definition, that is the moment mice touched the objects/mice in NOR test/social interaction test and the start of rotation (with a distinct body bend). To distinguish between rotation and running in rotational behavior test, we first analyzed behavior video in SMART v.3.0 software to get the onset time of rotation automatically, then we match it in the fiber photometry system.2+ traces were plotted using MATLAB program.The data were normalized (ΔF/F) by calculating the median signal across the recording period, subtracting this value from each data point, and dividing by the median signal. The area under the curve (AUCs) of ΔF/F in each defined time window were also calculated to quantify the change in fluorescence values induced by specific behavioral events. The heatmap and averaged CaAfter the behavioral experiments, the mice were anesthetized with isoflurane and transcardially perfused with 20 ml of 0.01 M phosphate buffered saline (pH 7.4), followed by 40 ml of 4% (w/v) paraformaldehyde (pH 7.4). For c-Fos staining, the mice were gently returned to their home cage and housed for 90 min without disturbing them before sacrificed. Brains were removed from the skull and then postfixed with 4% (w/v) paraformaldehyde at 4°C for 4 h. After cryoprotection with 30% (w/v) sucrose at 4°C for 48 h, the dehydrated brains were sectioned into 50 μm thick coronal slices using a freezing microtome . Representative slices of the ACC and BLA were chosen and counterstained with Hoechst  before imaging with a microscope  to confirm the locations of the virus injection and implanted optical fibers. For c-Fos staining, the brain slices from each group were washed with 0.01 M PBS, rinsed with blocking solution at room temperature for 2 h, and incubated with primary antibodies against c-Fos  overnight (4°C). The slices were washed with 0.01 M PBS the next day and probed with Alexa Fluor 488-conjugated anti-rabbit IgG  secondary antibodies at room temperature for 4 h. The microscopic fields of these slices were imaged using a confocal microscope objective (VS200) and processed with Imaris software .in situ hybridization histochemistry as previously described (2O2 in 0.1 M DEPC-PB at room temperature for 10 min and then treated with 0.3% (v/v) Triton X-100  in 0.1 M DEPC–PBS at room temperature for 20 min. Next, the sections were acetylated in an acetylation solution for 10 min at room temperature. After two washes with DEPC–PBS, the sections were incubated with hybridization buffer for 1 h at 58°C. The sections were then incubated with 1 μg/ml Vglut2 cRNA probe in hybridization buffer at 58°C for 18–20 h for hybridization. After two washes for 20 min each at 58°C, the sections were incubated with RNase buffer at room temperature for 5 min. Subsequently, the hybridized sections were treated with 10 μg/ml RNase A (Toyobo) in 10 mM Tris–HCl (pH 8.0), 0.5 M NaCl, and 1 mM EDTA for 30 min at 37°C. The sections were washed twice for 10 min each with 2 × SSC and 0.1% (w/v) NLS at 37°C and twice for 10 min each with 0.2 × SSC and 0.1% (w/v) NLS at 37°C. Sections were incubated with the blocking reagent at room temperature for 1 h. Then, the sections were incubated with a mixture of peroxidase-conjugated anti-digoxigenin antibodies  and rabbit anti-RFP antibodies  at room temperature overnight. After three washes, the sections were incubated with a mixture of Alexa Fluor 488-conjugated streptavidin to visualize the riboprobes  and Alexa Fluor 594-conjugated anti-rabbit IgG to amplify the virus  at room temperature for 4 h. Finally, all sections were incubated with Hoechst  for 10 min at room temperature. All sections were air-dried and cover-slipped with a fluorescent mounting medium.We performed escribed . The stat-test. Other data used non-parametric test. Parametric test contained two-tailed paired t-test and One-factor ANOVA here. Non-parametric test contained Mann–Whitney U test, Friedman’s M test and Kruskal–Wallis H test here. All statistical analyses were listed in the figure legends and were presented in p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001.All statistical data were shown as the mean ± SEM and were performed in Prism v.8.0  and statistical product service solutions (SPSS) v.21.0. At beginning, Shapiro–Wilk test was used to examine the normality test, and Levene’s test was used to examine homogeneity of variance test. Datasets that satisfied both the normality test and the homogeneity of variance test were analyzed with a parametric test. Datasets that satisfied the normality test but not the homogeneity of variance test were analyzed with a separate variance estimation p = 0.207; Control vs. MK-801 0.1 mg/kg: p < 0.001; Control vs. MK-801 0.2 mg/kg: p < 0.0001; Control vs. MK-801 0.4 mg/kg: p = 0.143). Rotational behavior was initiated 5 min after the MK-801 injection and peaked at approximately 10–15 min, and the effects lasted approximately 2 h (p < 0.0001). Therefore, we chose a concentration of 0.2 mg/kg MK-801 and 15 min injection interval for testing schizophrenia-like behaviors. In the open field test (OFT), the total distance traveled by the MK-801 group was increased significantly compared with that of the control group , which further indicated that MK-801 caused hyperactivity resembling the positive behavior of schizophrenia in mice. Patients with schizophrenia also show obvious apathy and social withdrawal  and bouts of the interaction  of MK-801-injected mice with the same homogeneous male mice were significantly reduced in the social interaction test . The preference score of time in the MK-801 group was significantly decreased compared with control mice , suggesting that their cognitive function was impaired. In addition, we also performed gait test to explore whether MK-801 caused other concomitant symptoms (such as dysfunction in locomotor coordination) in mice . Similar to the results of c-Fos staining, the dynamic calcium signals of ACC and BLA neurons were significantly increased within seconds of rotational behavior onset in the MK-801-injected mice, and few differences were observed in the control mice . In contrast, the density of c-Fos-positive cells in the ACC and BLA of the MK-801-injected mice was significantly decreased compared with that in the control mice in the social interaction test . The neuronal activities of the ACC and BLA in the MK-801 group also displayed an obvious decrease compared with the control group based on in vivo fiber photometry recording of the GCaMP6 signal . Finally, we examined c-Fos expression in these two brain regions after the NOR test, and the results showed no significant difference between the control mice and MK-801-injected mice . Changes in the calcium signals in the ACC and BLA of the MK-801 group were lower than those in the control group . For all mice in the c-Fos staining , indicating that the viral strategy was effective. However, in the positive behavior tests, neither the stereotypic rotational behavior nor the hyperactivity of the mice was altered by chemogenetic regulation  and bouts compared with the stranger mouse . Although a significant difference in interaction time and bouts were not observed between the Gi-MK-801 and GFP-MK-801 mice, the Gi-MK-801 group also showed a significant reduction compared with the Gq-MK-801 group, which may be related to the “floor effect.” Last, we found that the Gq-MK-801 group spent more time exploring the novel object after activating the BLA-ACC circuit by C21 injection in the NOR test . However, no significant difference was observed in the Gi-MK-801 mice  and GFP-MK-801 mice  after C21 was injected. The preference score of Gq-MK-801 mice was also significantly higher than that of Gi-MK-801 and GFP-MK-801 mice . Interestingly, we also found that inhibition of BLA-ACC pathway could reduce the social interaction time and time spent in novel objects but not rotational behavior in vehicle injected-mice /GFP/hM4Di (Gi)-WPRE-bGH pA viruses were bilaterally delivered into the BLA, and an AAV2/Retro-hSyn-NLS-Cre-P2A-mCherry virus was bilaterally injected into the ACC by stereotactic injection . This apgulation . In socigulation , the micted-mice . Gait abted-mice . Based oted-mice . Taken t2) and optical inhibition with yellow light  were applied to activate or inhibit the BLA-ACC projection neurons during the behavioral test . In the positive behavior tests, neither the stereotypic rotational behavior nor the hyperactivity of the mice was regulated by blue or yellow light stimulation  and bouts of interaction  were significantly increased when the light was on in the ChR2-MK-801 group compared with the light off phase. The social interaction time  and bouts  of ChR2 mice were also increased compared with those in the mCherry-MK-801 group during the light-on period. The yellow light stimulation did not change the interaction time  and bouts of interaction  in the NpHR-MK-801 mice, which may be related to the “floor effect.” The results of the NOR test were similar to that in the chemogenetic regulation, the activation of BLA-ACC projection neurons with blue light increased the time spent exploring the novel object  and the preference score  compared with the mCherry-injected mice. Although there was no statistical difference in the time spent exploring the novel object after blue light activation this pathway, there was an obvious increase trend in the MK-801 + ChR2 group . The finding that the time mice spent exploring novel objects after yellow light-mediated inhibition of the circuit was not altered compared with before light exposure and even showed an increasing trend is confusing. But in vehicle injected mice, we found that inhibition of BLA-ACC pathway could reduce the time spent exploring novel objects of mice, and we also observed that activation of BLA-ACC pathway could increase the social interaction time and time spent in novel objects but not rotational behavior . The results indicated that rotational behavior in the NOR test might influence the c-Fos staining. However, the results of fiber photometry in NOR test showed that the calcium signals in the ACC and BLA of the MK-801 group were lower than those in the control group, which indicated that rotational behavior in the NOR test did not influence the results of fiber photometry recording. To sum up, these results indicated that mice treated with MK-801 indeed showed lower neural activation in the ACC and BLA than the control group in the NOR test. These phenomena are easy to understand separately. As shown in our previous studies, the ACC and BLA are activated after stereotypic behaviors  and glutamate systems in these regions . An exceior test  and in tior test . Interesehaviors , and ACCehaviors . Other lehaviors . The relehaviors . Howeverin situ hybridization on postmortem tissue showed a reduction in the expression of the NR2A subunit on parvalbumin-positive interneurons  of the Fourth Military Medical University.XH, HL, WW, and SW: conceptualization. XH, YL, and HL: methodology. XH, YL, HL, JX, BT, ZT, and HD: investigation. XH and HL: writing—original draft. WW and SW: writing—review and editing, funding acquisition, resources, and supervision. All authors read and approved the final manuscript.